CN103834605B

CN103834605B - A kind of Abamectin producing bacterium and its preparation method and application

Info

Publication number: CN103834605B
Application number: CN201210477320.7A
Authority: CN
Inventors: 刘�文; 赵群飞
Original assignee: Shanghai Institute of Organic Chemistry of CAS
Current assignee: Shanghai Institute of Organic Chemistry of CAS
Priority date: 2012-11-21
Filing date: 2012-11-21
Publication date: 2018-09-21
Anticipated expiration: 2032-11-21
Also published as: CN103834605A

Abstract

The present invention relates to a kind of Abamectin producing bacteriums and its preparation method and application.Specifically, the aveD genes in the Abamectin producing bacterium of wild type are inactivated, elimination component A can be obtained, only generate the bacterial strain of B component；AveD genes and aveC genes are inactivated simultaneously, and cover the homologous gene of aveC on this basis（It is preferred that the meiC genes in Meiling-mycin biological synthesis gene cluster）, 95wt% or more is avermectin B2a in the mutant tunning of acquisition.Using product avermectin B2a as precursor, it is used directly for largely synthesizing the Avermectin B1a and ivermectin of high-purity.

Description

A kind of Abamectin producing bacterium and its preparation method and application

Technical field

The invention belongs to biotechnologies, in particular it relates to a kind of Abamectin producing bacterium and its preparation side Method and application.

Background technology

Avermectin (Avermectin) is one kind by gram-positive bacteria deinsectization streptomycete (Streptomycesavermectinius) the ten hexa-atomic macrolide antibiotics of one kind generated.Avermectin is a kind of high Effect, non-harmful biological pesticide, the anti-mite activity of desinsection with wide spectrum, and avermectin is low to people and animals' toxic side effect, in vivo Remain low, these features make avermectin have in terms of the treatment of domestic animal parasitic infection and control of agricultural pest extensively Application, be produce pollution-free food best medication.

In general, in abamectin fermenting process, component A is generated（A1a and A2a etc.）And B component（B1a and B2a Deng）(see Fig. 1) is divided into most effective composition with B1a groups, and B2a components are typically used as synthesizing another biological pesticide Yi Wei bacterium The precursor of element.Using the avermectin derivative Ivermectin HCL semi-synthetic as precursor（Ivermectin）And doractin (Doramectin) there is higher application value, the former with 23 double bonds by the 22 of B1a to be changed to singly-bound, and the latter is then by B1a 25 side-chain radicals be changed to cyclohexyl.

It needs to be purified from natural fermentation products in avermectin and ivermectin production at present, processing procedure is very Complexity, yield are low, it is also necessary to use the toxic solvents such as toluene.In addition, still no tunning only has the bacterial strain of B2a components.

Therefore there is an urgent need in the art to develop the clearly single Abamectin producing bacterium of fermentation component of high yield.

Invention content

It is an object of the invention to provide a kind of Abamectin producing bacteriums and its preparation method and application.

In the first aspect of the present invention, a kind of Abamectin producing bacterium is provided, in the tunning of the producing strains, The relative amount of avermectin B2a component meets Formulas I：

B/A≥35%

Formulas I

Wherein,

B is the quality of avermectin B2a component in tunning；

A is the quality that all avermectin components are total in tunning.

In another preferred example, B/A >=40%, more preferably >=50%, more preferably >=60%, more preferably >=70%, more preferably >= 80%, more preferably >=90%, more preferably >=95%, most preferably >=99%.

In another preferred example, the Abamectin producing bacterium is streptomyces.

In another preferred example, the Abamectin producing bacterium is deinsectization streptomycete (Streptomycesavermectinius)。

In another preferred example, the aveD genes in the genome of the producing strains are deactivated or aveD gene functions lose Mistake or aveD gene delections do not express aveD genes or do not express AveD albumen or the AveD albumen nonfunctionals of expression.

In another preferred example, the amino acid sequence of the AveD albumen such as SEQ ID NO：Shown in 1.

In another preferred example, in the genome of the producing strains aveC genes be deactivated or aveC gene functions lose, Or aveC gene delections；Or it does not express aveC genes or does not express AveC albumen or AveC albumen nonfunctionals；Also, the production Contain or express the homologous gene of aveC in the genome of raw bacterium；Or the homologous protein of AveC albumen is expressed in the producing strains.

In another preferred example, the amino acid sequence of the AveC albumen such as SEQ ID NO：Shown in 2.

In another preferred example, the homologous gene of the aveC is selected from the group：Milbemycin (Milbemycin) biology closes At gene milC, Neck Martin（nemadectin）Biosynthesis gene nemC, Meiling-mycin（meilingmycin）Biology closes At gene meiC.

In another preferred example, the homologous gene of the aveC is Meiling-mycin（meilingmycin）Biosynthesis base Because of meiC.

In another preferred example, the milC gene sources are in Harbin streptomycete (Streptomycesbingchenggensis)。

In another preferred example, the nemC gene sources are in cyaneogriseus streptomyces (Streptomycescyaneogriseus)。

In another preferred example, the meiC gene sources are in nanchang streptomycete Streptomycesnanchangensis。

In another preferred example, Meiling-mycin（meilingmycin）The protein sequence of biosynthesis gene meiC codings Such as SEQ ID NO：Shown in 3.

In another preferred example, the Abamectin producing bacterium is deinsectization streptomycete （Streptomycesavermectinius）MVL1003, deposit number are GCMCC No.6678.

In the second aspect of the present invention, provide the Abamectin producing bacterium described in first aspect purposes it be used as sending out Ferment produces the engineering bacteria of avermectin B2a component.

In the third aspect of the present invention, a kind of method preparing avermectin B2a component, including step are provided：

(a) Abamectin producing bacterium described in first aspect is cultivated, to obtain the fermentation production containing avermectin B2a Object；With

(b) the avermectin B2a component is isolated from tunning.

In the fourth aspect of the present invention, a kind of method producing avermectin B1a component, including step are provided：

(a) Abamectin producing bacterium described in first aspect is cultivated, to obtain the fermentation containing avermectin B2a component Product；

(b) the avermectin B2a component is isolated from tunning；With

(c) avermectin B2a of separation is converted to avermectin B1a component.

In another preferred example, step (c) conversion is by B2a component pyrohydrolysises, to obtain B1a components.

In the fifth aspect of the present invention, a kind of method producing ivermectin, including step are provided：

(b) the avermectin B2a component is isolated from tunning；With

(c) the avermectin B2a component of separation is converted to ivermectin.

In another preferred example, step (c) conversion is gone back to avermectin B2a with three-n-butyl stannanes Former and deprotection, obtains ivermectin.

In the sixth aspect of the present invention, a kind of method preparing Abamectin producing bacterium, including step are provided：

(1) to the genetic modification for going out bacterium germination and carrying out aveD genes and/or AveD albumen of production ivermectin, to obtain AveD genes inactivation or aveD gene functions are lost or aveD gene delections or do not express aveD genes or not in genome Express AveD albumen or the non-functional Abamectin producing bacterium of AveD albumen of expression；

Also, in going out in the tunning of bacterium germination for the production ivermectin, the relative amount of avermectin B2a component Meet Formula II：

B/A ＜ 35%

Formula II

Wherein,

B is the quality of avermectin B2a component in tunning；

A is the quality that all avermectin components are total in tunning.

In another preferred example, the method further includes step：

(2) bacterial strain obtained to step (1) carries out further genetic modification to aveC genes and/or AveC albumen, from And it obtains aveC genes inactivation or the forfeiture of aveC gene functions or aveC gene delections in genome or does not express aveC bases Cause does not express AveC albumen or the non-functional Abamectin producing bacterium of AveC albumen of expression；And

(3) bacterial strain obtained to step (2), covers the homologous gene or AveC homologous proteins of aveC.

In the seventh aspect of the present invention, provide a kind of assortment of genes, the assortment of genes with set out（Or it is original）'s The assortment of genes of production ivermectin is compared, and has one or more features selected from the group below：

(1) in the assortment of genes, aveD genes be deactivated or aveD gene functions lose or aveD gene delections or AveD genes are not expressed；Or

(2) in the assortment of genes, aveC genes are deactivated or aveC gene functions lose or aveC gene delections；Or AveC genes are not expressed；Also, contain or express the homologous gene of aveC.

In the seventh aspect of the present invention, a kind of carrier is provided, the base described in the 6th aspect is integrated on the carrier Because of combination.

In the eighth aspect of the present invention, a kind of host cell is provided, the host cell contains described in the 7th aspect Carrier or the host cell integral have the 6th aspect described in the assortment of genes.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Following drawings is for illustrating specific embodiments of the present invention, rather than limits and be defined by the claims The scope of the invention.

Fig. 1 shows the chemical constitution of 8 components of avermectin.

Fig. 2 shows different strains fermentation production HPLC analysis result；I is starting strain fermentation production HPLC analysis knot Fruit；II is the mutant strain mVL1001 fermentation production HPLC analysis results of aveD inactivations, only generates avermectin B component；III is aobvious Show, the fermentation production HPLC analysis result of mutant strain mVL1002, does not produce AVM hereinafter B1a completely after mVL1001 carries out aveC inactivations Component only generates micro B2a components；IV shows that the mutant strain mVL1003 that heterologous covering meiC is obtained in mVL1002 is sent out Ferment product HPLC analysis results only generate avermectin B2a one-component.

Specific implementation mode

The present inventor after extensive and in-depth study, provides a kind of AVM hereinafter of high yield avermectin B2a component for the first time Rhzomorph producing strains and its preparation and application.Specifically, the aveD genes in the Abamectin producing bacterium of wild type are inactivated, Elimination component A can be obtained, the bacterial strain of B component is only generated, to increase substantially the content of B2a components in tunning； AveD genes and aveC genes inactivate simultaneously, and cover the homologous gene of aveC on this basis（Meiling-mycin biosynthesis base Because of the meiC genes in cluster）, 95wt% or more is avermectin B2a in mutant tunning.By product avermectin B2a As synthesis precursor, it is used directly for the synthesis of high-purity Abamectin B1a and ivermectin.Complete this on this basis Invention.

Term

As used herein, term " more than " and " following " include this number, for example, " 90% or more " refer to >=90%, " 0.2% with Under " refer to≤0.2%.

Term " without/avermectin A or B1a component are not generated " or " substantially free of/avermectin A or B1a are not generated Component " is used interchangeably, and refers to content≤0.1wt% of avermectin A or B1a component in tunning, preferably≤0.01wt%, More preferably≤0.005wt% (such as 0wt%).

Bacterial strain

As used herein, term " starting strain of the present invention ", " wild strain of the present invention " or " present invention set out microorganism " It may be used interchangeably, all refer to deinsectization streptomycete StreptomycesavermectiniusATCC31267.

The starting strain of the present invention comes from American Type Culture collection warehousing (American type Culturecollection, ATCC), number ATCC31267.

Starting strain for the present invention includes not only deinsectization streptomycete StreptomycesavermectiniusATCC31267 further includes its derivative strain and the bacterial strain of other production avermectin.

Culture presevation

The deinsectization streptomycete of the present invention（Streptomyces avermectinius）MVL1003, October 18 in 2012 Day be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) (Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number CGMCCNo.6678.

Primer

As used herein, term " primer " refers to matching with template, can be with it under the action of archaeal dna polymerase Point carries out the general name of synthesis and the oligonucleotide acid of the DNA chain of template complementation.Primer can be natural RNA, DNA, can also It is any type of natural nucleotide.Primer can even is that non-natural nucleotide such as LNA or ZNA etc..

Primer " generally " (or " substantially ") and the special sequence of one on a chain in template are complementary.Primer is necessary It could start to extend with an abundant complementation of chain in template, but the sequence of primer need not be with the sequence complete complementary of template.Than Such as, at one 3 ', end and 5 ' ends of the primer of template complementation plus the preceding paragraph and the not complementary sequence of template, such primer are still big It is complementary with template in cause.As long as there is sufficiently long primer that can adequately be combined with template, non-fully complementary primer can also be with Template forms primer-template complex, to be expanded.

Construction recombination plasmid

It can easily be used according to gene cluster as described herein and its nucleotide sequence at external both ends, those skilled in the art The homologous recombination sequence of the present invention is made in various known methods.These methods are such as, but not limited to：PCR, DNA are artificial synthesized etc., Specific method can be found in J. Pehanorm Brookers,《Molecular Cloning:A Laboratory guide》.

Described " being operatively connected " or " being operably coupled to " refers to such a situation, i.e., linear DNA molecule is certain Part can adjust or control the activity of same linear DNA molecule other parts.For example, if promoter control sequence turns Record, then it is exactly to be operably coupled to coded sequence.

The present invention also provides a kind of shuttle vectors.In the preference of the present invention, shuttle vector can Think the pKC1139 as gene knockout and the pSET152 plasmids as expression.According to the restriction enzyme mapping of known carrier, ability Field technique personnel conventionally can be sheared and be spliced by restriction enzyme, the sequence of the present invention is inserted into suitable restricted The recombinant vector of the present invention is made in site.

Conversion

By the vector introduction host cell containing the coded sequence, a variety of known technologies of this field can be used, including But it is not limited to：Calcium phosphate precipitation, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction Method, alkali metal ion method.When host is prokaryotes such as Escherichia coli, can absorb the competent cell of DNA can give birth in index Harvest, uses CaCl after long-term₂Method processing, step used are generally well-known in the art.Another method is to use MgCl₂.If It needs, conversion can also be carried out with the method for electroporation.When host is eucaryote, following DNA transfection methods can be selected：Phosphoric acid Calcium coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, replicate the gene order of the present invention.It is thin according to host used Born of the same parents, culture medium used in culture can be selected from various conventional mediums.It is cultivated under conditions of suitable for host cell growth. After host cell growth is to cell density appropriate, inducing selection (such as temperature transition or chemical induction) with suitable method Cell is further cultured for a period of time by promoter.

The present invention also provides a kind of host cells, wherein the coded sequence containing the present invention.The host cell is excellent Choosing is prokaryotic cell, and it is that host methylates the E. coli of modification missing that one, which is suitable for the invention host cell, ET12567。

Homologous recombination

Homologous recombination (Homologus Recombination) refers to being happened at sister chromatid (non- Sisterchromatin the group again between) or on same chromosome between the DNA molecular containing homologous sequence or within molecule It closes.Homologous recombination needs the catalysis of a series of protein, as in prokaryote RecA, RecBCD, RecF, RecO, RecR etc.；And Rad51, Mre11-Rad50 etc. in eukaryotic cells.Homologous recombination reaction generally according to crossed molecular or Holiday structures (Holiday JunctureStructure).It is homologous between the stringent dependence DNA molecular of homologous recombination reaction Property.

Homologous recombination can be used for gene knockout.The technology path of gene knockout is as follows：

(1) structure recombination Zai Ti ﹔

(2) recombinant DNA is transferred to ﹔ in recipient cell karyon

(3) cell recombinated is screened with Selective agar medium.

In mammals, further include step (4)：By the cell recombinated be transferred to embryo make growth become turn base Because of animal, morphologic observation and molecular Biological Detection can also be carried out to transgenic animals.

Prepare the Abamectin producing bacterium of the present invention

The present invention provides a kind of methods preparing Abamectin producing bacterium, including step：

(1) to the genetic modification for going out bacterium germination and carrying out aveD genes and/or AveD albumen of production ivermectin, to obtain AveD genes inactivation or aveD gene functions are lost or aveD gene delections or do not express aveD genes or not in genome Express AveD albumen or the non-functional Abamectin producing bacterium of AveD albumen of expression；Also, in the production ivermectin Go out in the tunning of bacterium germination, the relative amount of avermectin B2a component meets Formula II：

B/A ＜ 35%

Formula II

Wherein,

B is the quality of avermectin B2a component in tunning；

A is the quality that all avermectin components are total in tunning.

In another preferred example, the method further includes step：

In the preference of the present invention, specifically, the method includes the steps：

(1) structure for homologous recombination carrier, the carrier successively contain be connected with each other by 0-2000bp bases, Contain the homology arm sequence positioned at aveD upstream and downstreams respectively；

(2) using the vector introduction of step (1) as starting strain, the bacterial strain for importing carrier is obtained, wherein described Starting strain, which can ferment, mainly generates avermectin A1a, A2a, B1a, B2a components；

(3) from the bacterial strain that step (2) obtains, the upstream homology arm sequence and downstream homology arm sequence is screened and is set out The bacterial strain of secondary homologous recombination has occurred in the genome of bacterial strain, to obtain the avermectin generation for only generating B1a and B2a components Bacterium；

(4) structure for homologous recombination carrier, the carrier successively contain be connected with each other by 0-2000bp bases, Contain the homology arm sequence positioned at aveC upstream and downstreams respectively；

(5) by the vector introduction step of step (4)（3）The bacterial strain of acquisition, wherein it is described the step of（3）The bacterium of acquisition Strain, which can ferment, generates Avermectin B1a, B2a components；

(6) from the bacterial strain that step (5) obtains, the upstream homology arm sequence and downstream homology arm sequence is screened and is set out The bacterial strain of secondary homologous recombination has occurred in the genome of bacterial strain；

(7) carrier of the structure for expressing aveC homologous genes, the carrier contain erythromycin promoter, complete successively AveC homologous gene fragments；

(8) by the vector introduction step of step (7)（6）The bacterial strain of acquisition；Obtain the Avermectin for only generating B2a components Plain producing strains.

In another preferred example, step (7) the aveC homologous genes are selected from：Milbemycin (Milbemycin) biology closes At gene milC, Neck Martin（nemadectin）Biosynthesis gene nemC, Meiling-mycin（meilingmycin）Biology closes At gene meiC.

In another preferred example, further include step after step (3)：Whether verification Abamectin producing bacterium aveD genes are sent out Raw point mutation.

In another preferred example, further include step after step (6)：Whether verification Abamectin producing bacterium aveC genes lack It loses.

In another preferred example, step (1), (4) described homology arm sequence are obtained with the method for PCR amplification.

In another preferred example, step (7) the complete aveC homologous gene fragments are obtained with the method for PCR amplification 's.

In another preferred example, step (1), the length of (4) described homology arm sequence are not particularly limited, preferably >= 15000bp。

In another preferred example, step (1), (4), (7) described carrier are shuttle plasmid, preferably Escherichia coli-chain Mould shuttle plasmid.

In another preferred example, step (1), (4), (7) described carrier also contain antibiotics resistance gene, preferably Ah Uncle draws mycin resistant gene.

In another preferred example, step (2), (5), the introduction method streptomycete DNA introductory techniques described in (8), preferably Engagement transfer introductory technique.

In another preferred example, the starting strain described in step (2) is streptomyces, preferably deinsectization streptomycete.

The assortment of genes

The present invention also provides a kind of assortment of genes, the assortment of genes with set out（Or it is original）Production ivermectin The assortment of genes compare, have one or more features selected from the group below：

The present invention also provides a kind of carrier, the above-mentioned assortment of genes is integrated on the carrier.

The present invention also provides a kind of host cell, the host cell contains above-mentioned carrier or the host is thin Born of the same parents are integrated with said gene combination.

In another preferred example, the host cell is bacillus subtilis or streptomycete, preferably streptomycete, most preferably It is deinsectization streptomycete.

Fermenting and producing avermectin B2a

The bacterial strain of the present invention can be used for preparing avermectin B2a or its salt or derivatives thereof by bioanalysis.Wherein, described Salt include (but being not limited to)：Hydrochloride, sulfate, phosphate, acetate, citrate, other organic carboxylates etc..

The avermectin B2a derivative includes (but being not limited to)：Ivermectin, doractin etc..

The present invention fermenting and producing avermectin B2a production method, other than producing bacterium difference, other conditions with it is existing The method of fermenting and producing is essentially identical in technology, such as to the purifying process of avermectin B crude products.The fermentation item of bacterial strain of the present invention Part is close with general streptomycete, i.e., in the culture medium of carbonaceous sources, nitrogen source and trace element, pH5.0-9.0 (preferably PH7.0-7.5 it) ferments with 20-45 DEG C (preferably 25-40 DEG C).In the present invention, " mycelium ", " zymotic fluid " or " culture solution " It can make growth to certain mycelia bulk concentration by the culture bacterial strain of the present invention under conditions of being suitble to growth to obtain.For Nutrient source in the culture medium of culture bacterial strain of the present invention is not particularly limited.Those skilled in the art can be according to well known skill Art selects suitable carbon source, nitrogen source and other nutrient sources.For example, carbon source can be starch, dextrin, glucose, fructose, sugarcane Sugar, glycerine, inositol, mannitol etc..Nitrogen source can be peptone, soy meal, soybean cake powder, meat extract, albumen powder, wheat skin, rice sugar, yeast Powder, corn steep liquor, ammonium salt and other organic matters or inorganic nitrogen-containing compound.In addition, can also be suitably added some nothings in culture medium Machine salt, such as sodium chloride, phosphate (such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate), manganese sulfate, ammonium sulfate, magnesium sulfate, carbonic acid The metal salts such as calcium.Various known conventional mediums usually can be used, such as LB agar mediums, nutrient agar, grape Sugar yeast cream agar medium and ox meat extract agar medium etc. to this bacterial strain carry out under inclined-plane solid culture and 4 DEG C of environment into The preliminary preservation of row.In a specific embodiment, there is consisting of (% expressions for cultivating the culture medium of bacterial strain of the present invention Mass/volume)：Soybean cake powder 2%, PEARLITOL 25C 2%, agar 2%.

However, it is understood by those of ordinary skill in the art that the invention is not limited in these the specific cultures enumerated herein Based formulas.

The conditions such as temperature, pH, gas liquid ratio, tank pressure, the rotating speed of bacterial strain in the culture present invention are not particularly restricted, As long as the condition is suitble to the growth of the bacterium.In some preferable embodiments, pH is preferably controlled between 6.5~8.0, training Foster temperature is preferably between 25~40 DEG C.It should be understood that the fermentation of the present invention can be continuously fermented, can also be Intermittent fermentation. In the preferable embodiment of the present invention, culture by the following method obtains zymotic fluid and mycelium：MS solid cultures Base（Soybean cake powder 2%, PEARLITOL 25C 2%, agar 2%）Upper 30 DEG C are cultivated 7 days.Cut about 1cm²The agar containing spore and mycelia Block accesses one grade fermemtation culture medium（Cornstarch 3%, bean cake powder 0.8%, groundnut meal 1%, yeast extract 0.4%, amylase 4 μ/g starch）, 30 DEG C, 200rpm cultivate 48 hours.Seed culture fluid is accessed into fermentation medium (jade by the inoculum concentration of 10% volume Rice starch 14%, bean cake powder 2%, yeast powder 1%, sodium molybdate 0.22%, ammonium sulfate 0.5%), 30 DEG C, 200rpm are cultivated 8 days.

Main advantages of the present invention include：

(1) the bacterial strain performance favorable reproducibility of genetic modification, modified Abamectin producing bacterium are carried out with the method for the present invention Inheritance stability is not easy to be mutated, and avermectin A and B1a component is no longer generated in tunning, and 95wt% or more products are Avermectin Plain B2a；

(2) using product avermectin B2a as precursor, high-purity Abamectin B1a and ivermectin can be synthesized, greatly Ground simplifies the production technology of ivermectin, improves production efficiency, significantly reduces production cost；

(3) engineering bacteria fermentation component of the present invention after genetic engineering is modified is clearly single, in rear extraction process not The toxic solvents such as toluene are recycled, the safety of working condition and production is significantly improved；Reduce other components as useless The waste of biomass caused by gurry is abandoned and thus caused environmental pollution；

(4) due to the purifying of avermectin component, the biological pesticide thus produced contains inefficient structure similar to impurity The reduction of object can greatly delay the generation of insect drug resistance during dispenser.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as Sambrook et al., molecular cloning：Laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1

Avermectin biosynthesis gene aveD inactivations（AveD point mutation）The structure of bacterial strain

1. recombinant plasmid of the structure for aveD point mutation

The primer sequence that PCR clones aveD upstream region of gene DNA fragmentations in avermectin biological synthesis gene cluster is as follows：

Primer 1：5’-TATGAA TTCCCT CGT CGA GGT GGC CGA G-3 ' (underscore is the sites EcoRI) (SEQ ID NO：4)

Primer 2：5’-TTAAAG CTTACC GCA GCC GAC GTC CAG G-3 ' (underscore HindIII) (SEQ ID NO：5)

The primer sequence that PCR clones aveD downstream of gene DNA fragmentations in avermectin biological synthesis gene cluster is as follows：

Primer 3：5’-TTA AAG CTTAAG CCG GCG GTG CGG CTC G-3 ' (underscore HindIII) (SEQ ID NO：6)

Primer 4：5’-TTATCT AGATCC TCA CCC TTT CCC CCG GC-3 ' (underscore XbaI) (SEQ ID NO：7)

With the Abamectin producing bacterium deinsectization streptomycete StreptomycesavermectiniusATCC31267's of extracting Total DNA is template, respectively with above-mentioned two group-specific primers to the upstream and downstream sequence of avermectin biosynthesis gene aveD into Row PCR amplification obtains the amplified fragments of the amplified fragments of the 2.0kb of gene cluster upstream and the 1.8kb in downstream.By the two segments After carrying out EcoRI-HindIII and HindIII-XbaI digestions respectively, it is (a kind of temperature sensitive big to be connected into commercially available pKC1139 Enterobacteria-streptomycete shuttle plasmid, US5,955,319) the sites EcoRI-XbaI, to obtain the recombination matter of aveD point mutation Grain pVL1001.AveD is guarded to the 79th glycine in the regions DxGxGxG（Gly）Sport leucine（Leu）, simultaneously will 78th serine（Ser）Sport lysine（Lys）, to form the sites HindIII.

By the conventional E. coli DH5 α of recombinant plasmid pVL1001 conversions, picking monoclonal colonies are cultivated in LB Overnight incubation in liquid (containing 100 μ g/ml of A Baila mycins antibiotic), until bacterium solution is denseer.Recombinant plasmid is extracted, through digestion verification Afterwards, business sequencing is carried out, the results show that recombinant shuttle plasmid structure is correct.

The construction and screening of 2.aveD point mutation mutant strains mVL1001

Correct plasmid pVL1001 will be verified and be transformed into the E. coli E that the conventional modification that methylates lacks Plasmid pVL1001 is imported AVM hereinafter by T12567 (referring to US7,326,782 and US7,105,491), the method by engaging transfer In rhzomorph producing strains Streptomyces avermectiniusATCC31267.

The joint element with A Baila chloramphenicol resistances is selected, joint element is placed in 37 DEG C of growths, is made in plasmid pVL1001 With the homologous fragment on chromosome homologous recombination occurs for aveD upstream region of gene or segments downstream, then by 37 DEG C of well-growns TSB fluid nutrient medium (culture medium be purchased from Sigma-Aldrich company) of the zygote access without any antibiotic in, 30 It is cultivated at DEG C in the TSB fluid nutrient mediums for taking a small amount of bacterium solution renewed vaccination to enter antibiotic-free two days later, after so repeating 2 wheels, Bacterium solution is crossed on MS solid mediums, selects the single bacterium colony for losing A Baila chloramphenicol resistances.

Due to being cultivated under the conditions of antibiotic-free, importing about 3-5% in the plasmid of streptomycete can be secondary same with genome generation Source recombinates, to lose A Baila chloramphenicol resistances.For the single bacterium colony for the forfeiture A Baila chloramphenicol resistances selected, DNA is extracted, is used PCR product carries out digestion method and determines genotype.

The primer used is：

Primer 5：5’-CCC CGC CGT CCA TCC TCT G-3’(SEQ ID NO：8)；

Primer 6：5’-TAC CGC GGC CGC ACT CAC G-3’(SEQ ID NO：9)；

The 2.01kb PCR products of wild type cannot be by HindIII digestions, and correctly aveD point mutation mutant strains 2.01kb PCR products can be identified, inventor by HindIII digestions at 0.59kb and 1.42kb through PCR product digestion and sequencing The genetic engineering transformation bacterial strain for successfully constructing correct aveD point mutations, is named as mVL1001.

Embodiment 2

The structure of the bis- mutant strains of avermectin biosynthesis gene aveC aveD

1. the plasmid that structure aveC is knocked out with frame

The primer sequence that PCR clones aveC upstream region of gene DNA fragmentations in avermectin biological synthesis gene cluster is as follows：

Primer 7：5’-TTAGAA TTCCAT CAC GCT GCT GGA CTT CG-3 ' (underscore is the sites EcoRI) (SEQ ID NO：10)

Primer 8：5’-ACG CCT GCA GGG CCA GAA ACA G-3 ' (underscore PstI) (SEQ ID NO：11)

The primer sequence that PCR clones aveC downstream of gene DNA fragmentations in avermectin biological synthesis gene cluster is as follows：

Primer 9：5’-TATCTG CAGGTG AAT GCC GTG ATG TTC CTC-3 ' (underscore PstI) (SEQ ID NO：12)

Primer 10：5’-TATTCT AGATGC TCT CCT CCA CCA CAT CCT C-3 ' (underscore XbaI) (SEQ ID NO：13)

With the Abamectin producing bacterium deinsectization streptomycete StreptomycesavermectiniusATCC31267's of extracting Total DNA is template, respectively with above-mentioned two group-specific primers to the upstream and downstream sequence of avermectin biosynthesis gene aveC into Row PCR amplification obtains the amplified fragments of the amplified fragments of the 1.87kb of gene cluster upstream and the 1.9kb in downstream.By the two pieces After section carries out EcoRI-PstI and PstI-XbaI digestions respectively, it is connected into a kind of pKC1139 (temperature sensitive Escherichia coli-strepto-s Bacterium shuttle plasmid, US5,955,319) the sites EcoRI-XbaI, to obtain the recombinant plasmid that aveC is knocked out with frame pVL1002.Wherein aveC lacks 756bp with frame.

By the conventional E. coli DH5 α of recombinant plasmid pVL1002 conversions, picking monoclonal colonies are cultivated in LB Overnight incubation in liquid (containing 100 μ g/ml of A Baila mycins antibiotic), until bacterium solution is denseer.Recombinant plasmid is extracted, through digestion verification Afterwards, business sequencing is carried out, the results show that recombinant shuttle plasmid structure is correct.

Construction and screenings of the 2.aveC with frame knockout mutant strain mVL1002

Correct plasmid pVL1002 will be verified and be transformed into the E. coli that the conventional modification that methylates lacks ET12567 (reference can be made to US7,326,782 and US7,105,491), the method by engaging transfer, plasmid pVL1002 is imported In bacterial strain mVL1001 according to the aveD point mutation of the structure of embodiment 1.The joint element with A Baila chloramphenicol resistances is selected, it will Joint element is placed in 37 DEG C of growths, makes homologous on aveC upstream region of gene or the segments downstream and chromosome in plasmid pVL1002 Homologous recombination of Duan Fasheng, then by the 37 DEG C of well-grown TSB Liquid Cultures of zygote access without any antibiotic In base (being purchased from Sigma-Aldrich companies), is cultivated at 30 DEG C and a small amount of bacterium solution renewed vaccination is taken to enter antibiotic-free two days later In TSB fluid nutrient mediums, after so repeating 2 wheels, bacterium solution is crossed on MS solid mediums, select and lose A Baila mycins The single bacterium colony of resistance.

Due to being cultivated under the conditions of antibiotic-free, importing about 3-5% in the plasmid of streptomycete can be secondary same with genome generation Source recombinates, to lose A Baila chloramphenicol resistances.For the single bacterium colony for the forfeiture A Baila chloramphenicol resistances selected, DNA is extracted, is used The method of PCR determines genotype.

Primer 11：5’-CCC CGC CGT CCA TCC TCT G-3’(SEQ ID NO：14)；

Primer 12：5’-TAC CGC GGC CGC ACT CAC G-3’(SEQ ID NO：15)；

The mutant strain of starting strain aveD point mutation obtains 1.19kb PCR products, and the correctly bis- mutant bacterias of aveCaveD Strain obtains 0.43kb PCR products.PCR product identifies that inventor successfully constructs correct aveCaveD Gene Doubles mutation through sequencing Genetic engineering be transformed bacterial strain, be named as mVL1002.

Embodiment 3

The structure of the heterologous covering bacterial strains of meiC

1. building the plasmid of the heterologous covering of meiC

The primer sequence that PCR clones Meiling-mycin biosynthesis gene meiC is as follows：

Primer 13：5’-TATGGA TCC(underscore is BamHI to CTG CGC AAT AGG CTC ACC AC-3 ' Point) (SEQ ID NO：16)

Primer 14：5’-TATTCT AGACGG GAG GTG TCT ACA AGT GC-3 ' (underscore is the sites XbaI) (SEQ ID NO：17)

Using the total DNA of the Strain Producing Meilingmycin nanchang streptomycete Streptomyces nanchangensis of extracting as mould Plate, with above-mentioned a pair of of specific primer to the PCR amplification that carries out of Meiling-mycin biosynthesis gene meiC, obtaining 1.7kb includes The PCR product of complete meiC genetic fragments.After PCR product is carried out BamH-XbaI digestions, it is red mould to be connected into containing for conventional commercial The BamH- of pSET152 that element starts (integrated Escherichia coli-streptomycete shuttle plasmid, reference can be made to US1,171,583) a kind of The sites XbaI, to obtain the recombinant plasmid pVL1003 for the heterologous covering of meiC.

By the conventional E. coli DH5 α of recombinant plasmid pVL1003 conversions, picking monoclonal colonies are cultivated in LB Overnight incubation in liquid (containing 100 μ g/ml of A Baila mycins antibiotic), until bacterium solution is denseer.Recombinant plasmid is extracted, through digestion verification Afterwards, sequencing result proves, recombinant shuttle plasmid structure is correct.

The construction and screening of the heterologous covering bacterial strain mVL1003 of 2.meiC

Correct plasmid pVL1003 will be verified and be transformed into the E. coli that the conventional modification that methylates lacks ET12567 (referring to US7,326,782 and US7,105,491), the method by engaging transfer press plasmid pVL1003 importings According in the bacterial strain mVL1001 of the bis- mutation of aveCaveD of the structure of embodiment 2.The joint element with A Baila chloramphenicol resistances is selected, Joint element is placed in 30 DEG C of growths, that is, obtains the engineering strain of the heterologous covering of meiC, is named as mVL1003.

Deinsectization streptomycete（Streptomyces avermectinius）MVL1003 was deposited on October 18th, 2012 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) (city of BeiJing, China), preserving number CGMCC No.6678。

Embodiment 4

The fermentation and detection of recombinant bacterial strain

1. seed activation and culture

The spore of recombinant bacterial strain mVL1001, mVL1002 and mVL1003 that -80 DEG C preserve are coated on MS solid mediums （Soybean cake powder 2%, PEARLITOL 25C 2%, agar 2%）On, 30 DEG C are cultivated 7 days.Cut about 1cm²The agar block containing spore and mycelia Access one grade fermemtation culture medium (cornstarch 3%, bean cake powder 0.8%, groundnut meal 1%, yeast extract 0.4%, 4 μ of amylase/g Starch), 30 DEG C, 200rpm cultivate 48 hours, obtain in next step test seed culture fluid.

2. expanding culture

Seed culture fluid is accessed into fermentation medium (cornstarch 14%, bean cake powder 2%, yeast by the inoculum concentration of 10% volume Powder 1%, sodium molybdate 0.22%, ammonium sulfate 0.5%), 30 DEG C, 200rpm are cultivated 8 days.Zymotic fluid is harvested, is obtained containing avermectin The crude product of B2a.Cord blood is detected for yield.

3. tunning detects

Product is detected to prepare：The methanol that 4 times of volumes are added in zymotic fluid impregnates, ultrasound 15 minutes, centrifuging and taking supernatant, by methanol 3 times of dilution is later used to HPLC and LC-MS detections.

HPLC and LC-MS analyzes testing conditions：

Instrument：Agilent 1100HPLC systems；

Pillar：Agilent ZORBAX SB-C18 columns (4.6x250mm)；

Detection wavelength：UV=245nm；

Flow velocity：1ml/min；

Mobile phase：A=H₂O；B=methanol；

Use 85%B/15%A perseverances condition of gradient elution for elution time 20min.

As a result：Different strains fermentation production HPLC analysis result is as shown in Figure 2.

I is starting strain fermentation production HPLC analysis result, generates 8 components of avermectin, and wherein B2a components account for about always The 30% of component.

II is the mutant strain mVL1001 fermentation production HPLC analysis results of aveD point mutation, only generates avermectin B groups Point, wherein B2a components account for about the 42% of total component.

III is the fermentation production HPLC analysis result of the mutant strain mVL1002 after mVL1001 carries out aveC knockouts, completely AVM hereinafter B1a components are not produced, only generate micro B2a components.

IV is the mutant strain mVL1003 fermentation production HPLC analysis results that heterologous covering meiC is obtained in mVL1002, only Avermectin B2a one-component is generated, wherein B2a components account for about 95% or more of total component.

The result shows that inventor obtains the fermentation of strain by carrying out genetic engineering transformation to wild type deinsectization streptomycete Product 95wt% or more is avermectin B2a component, no longer generates avermectin component A and B1a components.

Embodiment 5

Avermectin B2a Composition Active detects

Test method：Cotten aphid insecticidal activity is tested：Clean fresh 1 small pieces of melon leaf are taken, with small size writing brush by size, body The consistent cotton aphid of color (Aphis gossypii Glover) selects 30 thereon, and blade is immersed liquid together with test worm It is taken out after middle 2s, liquid extra around cotton aphid is exhausted with blotting paper immediately, blade is put into the plastic cup of 50ml, cup Mouth is locked with rubber band with gauze and is escaped to prevent aphid, test worm is put into the recovery room of (27 ± 1) DEG C relative humidity 85%, 16h Inspection result afterwards.Death standard is gently to touch polypide with small size writing brush, and test worm is motionless and is considered as death without any reactor, The percentage of total borer population is accounted for as the death rate using dead borer population.Clear water control is set simultaneously, and school is calculated by Abbott formula with clear water control The positive death rate.0.50,1.50 and 2.50mg/L, tri- mass concentrations are set per medicament, are repeated 2~3 times under each mass concentration.

The result shows that：Avermectin B2a prepared by the present invention has good bioactivity.

Embodiment 6

Avermectin B2a component is converted into the experiment of other compounds as precursor

The method of avermectin B2a component synthesizing activity more preferably B1a components：Pass through t-butyldimethylsilyloxy base The alkylation that chloroacetic chloride carries out B2a 4 " and 5-OH base selectivity is protected, with 0-4- aminomethyl phenyls chloro-formate pair 23- OH bases are alkylated, and obtained alkylate is hydrolyzed 1 hour for 200 DEG C in 1,2,4- trichloro-benzenes, obtains C22-C23 At the compound of double bond.The derivative of 4 " and 5 double hydroxyacetyl modifications, last methanol are obtained after being handled with p- toluenesulfonic acids Sodium-methanol（0.2M, 60 minutes, 18 DEG C）Processing obtains activity more preferably B1a components.

The method that avermectin B2a component synthesizes ivermectin：Pass through t-butyldimethylsilyloxy base chloroacetic chloride pair B2a 4 " and 5-OH base carries out the alkylation protection of selectivity, is carried out with 23-OH bases of 0-4- aminomethyl phenyls chloro-formate pair Alkylation will obtain intermediate using toluene as solvent, and azodiisobutyronitrile carries out 1 hour back flow reaction as catalyst, obtains The product of C23 deoxidations, by obtaining 4 " and 5 pairs with P-TSOH x H20 processing in methyl alcohol immediately after TLC separation The positions the C23 deoxidation derivative of glycolic acid esters modification, finally uses sodium methoxide-methanol（0.2M, 60 minutes, 18 DEG C）Processing obtains her Tie up rhzomorph.

The result shows that：Avermectin B2a prepared by the present invention can successfully carry out subsequent Synthesis.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims

1. a kind of Abamectin producing bacterium, which is characterized in that the producing strains have following characteristics：

(a) the aveD genes in the genome of the producing strains are deactivated or aveD gene functions lose or aveD genes lack It loses or does not express aveD genes or do not express AveD albumen or the AveD albumen nonfunctionals of expression；

(b) aveC genes are deactivated in the genome of the producing strains or aveC gene functions lose or aveC gene delections； Or it does not express aveC genes or does not express AveC albumen or AveC albumen nonfunctionals；

(c) contain or express the homologous gene of aveC in the genome of the producing strains；And the homologous gene of the aveC is Meiling-mycin (meilingmycin) biosynthesis gene meiC；With

(d) in the tunning of the producing strains described in, the relative amount of avermectin B2a component meets Formulas I：

B/A >=35%

Formulas I

Wherein,

B is the quality of avermectin B2a component in tunning；

A is the quality that all avermectin components are total in tunning；

Also, the producing strains are deinsectization streptomycete (Streptomyces avermectinius).

2. Abamectin producing bacterium as described in claim 1, which is characterized in that B/A >=40%.

3. Abamectin producing bacterium as described in claim 1, which is characterized in that B/A >=95%.

4. Abamectin producing bacterium as described in claim 1, which is characterized in that the amino acid sequence of the AveD albumen is such as SEQ ID NO：Shown in 1.

5. Abamectin producing bacterium as described in claim 1, which is characterized in that the amino acid sequence of the AveC albumen is such as SEQ ID NO：Shown in 2.

6. Abamectin producing bacterium as described in claim 1, which is characterized in that the meiC gene sources are in Nanchang strepto- Bacterium (Streptomyces nanchangensis).

7. Abamectin producing bacterium as described in claim 1, which is characterized in that the Meiling-mycin (meilingmycin) The protein sequence such as SEQ ID NO of biosynthesis gene meiC codings：Shown in 3.

8. Abamectin producing bacterium as described in claim 1, which is characterized in that the production bacterium is deinsectization streptomycete (Streptomyces avermectinius) mVL1003, deposit number are CGMCC No.6678.

9. the purposes of the Abamectin producing bacterium described in claim 1, which is characterized in that it is used as fermenting and producing Avermectin The engineering bacteria of plain B2a components.

10. a kind of method preparing avermectin B2a component, which is characterized in that including step：

(a) Abamectin producing bacterium described in claim 1 is cultivated, to obtain the tunning containing avermectin B2a；With

(b) the avermectin B2a component is isolated from tunning.

11. a kind of method producing avermectin B1a component, which is characterized in that including step：

(a) Abamectin producing bacterium described in claim 1 is cultivated, to obtain the fermentation production containing avermectin B2a component Object；

(b) the avermectin B2a component is isolated from tunning；With

(c) avermectin B2a of separation is converted to avermectin B1a component.

12. method as claimed in claim 11, which is characterized in that step (c) it is described conversion be by B2a component pyrohydrolysises, To obtain B1a components.

13. a kind of method producing ivermectin, which is characterized in that including step：

(b) the avermectin B2a component is isolated from tunning；With

(c) the avermectin B2a component of separation is converted to ivermectin.

14. method as claimed in claim 13, which is characterized in that step (c) conversion is with three-n-butyl stannanes Avermectin B2a is restored and is deprotected, ivermectin is obtained.

15. a kind of method preparing Abamectin producing bacterium, which is characterized in that including step：

(1) to the genetic modification for going out bacterium germination and carrying out aveD genes and/or AveD albumen of production ivermectin, to obtain gene AveD genes inactivation or aveD gene functions are lost or aveD gene delections or do not express aveD genes or do not express in group AveD albumen or the non-functional Abamectin producing bacterium of AveD albumen of expression；

(2) bacterial strain obtained to step (1) carries out further genetic modification, to obtain to aveC genes and/or AveC albumen In genome aveC genes inactivation or aveC gene functions lose aveC gene delections or do not express aveC genes or AveC albumen or the non-functional Abamectin producing bacterium of AveC albumen of expression are not expressed；And

(3) bacterial strain obtained to step (2), covers the homologous gene of aveC；And the homologous gene of the aveC is that Meiling Hill is mould Plain (meilingmycin) biosynthesis gene meiC；

Also, meet in the relative amount of going out in the tunning of bacterium germination for the production ivermectin, avermectin B2a component Formula II：

B/A >=35%

Formula II

Wherein,

B is the quality of avermectin B2a component in tunning；

A is the quality that all avermectin components are total in tunning；

16. a kind of assortment of genes, which is characterized in that the assortment of genes phase of the assortment of genes and the production ivermectin to set out Than having one or more features selected from the group below：

(1) in the assortment of genes, aveD genes are deactivated or aveD gene functions are lost or aveD gene delections or not table Up to aveD genes；And

(2) in the assortment of genes, aveC genes are deactivated or aveC gene functions lose or aveC gene delections；Or not table Up to aveC genes；Also, contain or express the homologous gene of aveC；And the homologous gene of the aveC is Meiling-mycin (meilingmycin) biosynthesis gene meiC；

17. a kind of carrier, which is characterized in that integrate the assortment of genes described in claim 16 on the carrier.

18. a kind of host cell, which is characterized in that the host cell contains the carrier or described described in claim 17 The assortment of genes had the right described in requirement 16 of host cell integral.